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Anti-hepatitis B virus active constituents from Swertia chirayita
FITOTE-03069; No of Pages 8 Fitoterapia xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Fitoterapia
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Anti-hepatitis B virus active constituents from Swertia chirayita
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journal homepage: www.elsevier.com/locate/fitote
Ning-Jia Zhou a,c, Chang-An Geng a, Xiao-Yan Huang a, Yun-Bao Ma a, Xue-Mei Zhang a, Ju-Le Wang b, Ji-Jun Chen a,⁎ a b c
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China Medical School of Tibet University, Lhasa 850000, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China
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Article history: Received 2 September 2014 Accepted in revised form 10 November 2014 Available online xxxx
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Keywords: Swertiachiralatone A Swertiachoside A Swertiachirdiol A Swertiachoside B Anti-hepatitis B virus activity Swertia chirayita
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1. Introduction
36 37
Swertia chirayita belonging to the family Gentianaceae is distributed throughout the temperate Himalaya area (Kashmir to Bhutan, and Kashia hills) between 1200 and 3000 m above sea level. The whole plant is widely used by local people for the treatment of hepatitis, inflammation and digestive diseases [1]. Previous studies on this species led to the isolation of twenty-one xanthones, eleven triterpenoids, five secoiridoids, as well as eight other compounds including alkaloids, phenolic compounds and steroids [2–18]. Our previous investigation on Swertia mileensis, the congener plant of S. chirayita, resulted in a series of novel lactones with unprecedented structures and promising antihepatitis B virus (anti-HBV) activity [19–22]. According to our preliminary bioassay in vitro, the 50% EtOH–H2O extract of S. chirayita could inhibit the secretions of hepatitis B surface
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41 42 43 44Q3 45 46 47 48 49
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Four new compounds swertiachiralatone A (1), swertiachoside A (2), swertiachirdiol A (3) and swertiachoside B (4), together with twenty-six known ones were isolated from the ethanol extract of Swertia chirayita. Their structures were elucidated by extensive spectroscopic analyses (1D- and 2D-NMR, HRESIMS, UV, IR and [α]D). All compounds were evaluated for anti-hepatitis B virus (anti-HBV) activities on HepG 2.2.15 cells line in vitro, of which compounds 14 and 19 showed inhibitory activity on hepatitis B surface antigen (HBsAg) secretion with IC50 values of 0.31 ± 0.045 and 1.49 ± 0.033 mM; compounds 14 and 28 exhibited activity against hepatitis B e antigen (HBeAg) secretion with IC50 values of 0.77 ± 0.076 and 5.92 ± 1.02 mM; and eight compounds (8,9,13,14,24–26,29) possessed activity against HBV DNA replication with IC50 values of 0.07–0.33 mM. In particular (+)-cycloolivil-4′-O-β-D-glucopyranoside (14) exhibited inhibition not only on the secretions of HBsAg and HBeAg with IC50 values of 0.31 ± 0.045 mM (SI = 4.29) and 0.77 ± 0.076 mM (SI = 1.75), respectively, but also on HBV DNA replication with an IC50 value of 0.29 ± 0.034 mM (SI = 4.66). © 2014 Published by Elsevier B.V.
⁎ Corresponding author. Tel.: +86 871 65223265; fax: +86 871 65227197. E-mail address: [email protected] (J.-J. Chen).
antigen (HBsAg) and hepatitis B e antigen (HBeAg) with IC50 values of 1.08 ± 0.23 mg/mL (SI N 2.0) and 0.11 ± 0.031 mg/mL (SI N 19.6), respectively, as well as HBV DNA replication with an IC50 value of 0.17 ± 0.025 mg/mL (SI = 12.9). However, the components responsible for anti-HBV activity in S. chirayita were still unclear. In order to clarify the active constituents, bioassay-guided fractionation of the 50% EtOH–H2O extract resulted in four new compounds (Fig. 1), swertiachiralatone A (1), swertiachoside A (2), swertiachirdiol A (3) and swertiachoside B (4), together with twenty-six known ones, isoorientin (5) [23], 2-C-β-D-glucopy-ranosyl-1, 3, 7trihydroxyxanthane (6) [24], mangiferin (7) [25], 8-O-[β-Dxylopyranosyl-(1 → 6)-β-D-glucopyranosyl]-1, 7-dihydroxyl3-methoxyxanthone (8) [26], 8-O-[β-D-xylopyranosyl-(1 → 6)β-D-glucopyranosyl]-1-hydroxyl-3, 7-dimethoxy-xanthone (9) [27], 1-O-β-D-glucopyranosyl-3, 5, 8-trihydroxyxanthone (10) [28], 7-O-[β-D-xylopyranosyl-(1 → 2)-β-D-xylopyranosyl]1, 8-dihydroxy-3-methoxyxanthone (11) [29], 1, 5, 8trihydroxyl-3-methoxyxanthone (12) [30], 1-hydroxy-3, 7-
http://dx.doi.org/10.1016/j.fitote.2014.11.011 0367-326X/© 2014 Published by Elsevier B.V.
Please cite this article as: Zhou N-J, et al, Anti-hepatitis B virus active constituents from Swertia chirayita, Fitoterapia (2014), http:// dx.doi.org/10.1016/j.fitote.2014.11.011
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N.-J. Zhou et al. / Fitoterapia xxx (2014) xxx–xxx
spectra were taken on a Shimadzu UV-2401PC spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were measured on a Bio-Rad FTS-135 spectrometer (Bio-Rad, Hercules, California, USA) with KBr pellets. 1D and 2D-NMR spectra were recorded on Bruker AM-400 or DRX-500 spectrometers (Bruker, Bremerhaven, Germany). Silica gel (200–300 mesh) for column chromatography was obtained from Qingdao Makall Chemical Company (Makall, Qingdao, China). Semi-preparative HPLC was conducted on Waters Alliance 2695 liquid chromatography with a ZORBAX SB-C18 (5 μm, 9.4 × 250 mm) column (Agilent, USA). Sephadex LH-20 (20–150 μm, Pharmacia, Sweden) and ODS (45–70 μm, Merck, Germany) were used for column chromatography. Fractions were monitored by thin-layer chromatography (TLC), and spots were visualized by heating silica gel plates after sprayed with 10% H2SO4 in EtOH.
104 105
2.2. Plant material
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2. Experimental
100
2.1. General experimental procedures
101 102
Mass spectra were run on LCMS-IT-TOF spectrometer (Shimadzu, Kyoto, Japan). Optical rotations were obtained on a JASCO model 1020 polarimeter (Horiba, Tokyo, Japan). UV
84 85 86 87 88 89 90 91 92 93 94 95 96 97
103
C
E
R
R
83
O
81 82
C
79 80
N
77 78
U
75 76
D
99
73 74
2.3. Extraction and isolation
T
98
dimethoxyxanthone (13) [31], (+)-cycloolivil-4′-O-β-Dglucopyranoside (14) [32], 8′-α-hydroxyl-lariciresinol-4-Oβ-D-glucopyranoside (15) [33], 8′-α-hydroxyllariciresinol4′-O-β-D-glucopyranoside (16) [34], 3, 4′-dihydroxy-3′methoxypropiophenone 3-O-β-D-glucopyranoside (17) [35], epi-syringaresinol-4″-O-β-D-glucopyranoside (18) [36], syringaresinol 4″-O-β-D-glucopyranoside (19) [37], 6′-Oβ-D-glucopyranosylgentiopicroside (20) [38], 6′-O-β-Dglucopyranosylsweroside (21), djalonenol (22) [39], swerbimalactone B (23), 3β-hydroxy-11-oxo-olean-12-enyl3-palmitate (24) [40], erythrodiol-3-O-palmitate (25) [41], olean-12-ene-28-carboxy-3β-hexadecanoate (26) [42], cholest4-en-3-one (27) [43], 3, 3′, 5-trihydroxybiphenyl (28) [44], bridelionoside B (29) [45], and (6R, 7E, 9R)-9-hydroxymegasigma-4, 7-dien-3-one-9-O-β-D-glucopyranoside (30) [46]. All the known compounds were isolated from this plant for the first time. Isolates 1–30 were evaluated for their antiHBV activities on HepG 2.2.15 cells line in vitro, of which compounds 14 and 19 showed inhibitory activity on HBsAg secretion with IC50 values of 0.31 ± 0.045 and 1.49 ± 0.033 mM; compounds 14 and 28 showed activity against HBeAg secretion with IC50 values of 0.77 ± 0.076 and 5.92 ± 1.02 mM; and eight compounds (8,9,13,14,24–26,29) exhibited activity against HBV DNA replication with IC50 values of 0.07–0.33 mM. In particular (+)-cycloolivil-4′-O-β-Dglucopyranoside (14) exhibited inhibition not only on the secretions of HBsAg and HBeAg with IC50 values of 0.31 ± 0.045 mM (SI = 4.29) and 0.77 ± 0.076 mM (SI = 1.75), respectively, but also on HBV DNA replication with an IC50 value of 0.29 ± 0.034 mM (SI = 4.66).
71 72
The whole plant of Swertia chirayita (Roxb. ex Fleming) H. Karst. was collected in Nepal, in September 2008 and identified by Prof. Ju-Le Wang, Medical School of Tibet University. A voucher specimen (No. 2008-11-30) was deposited at the Laboratory of Antivirus and Natural Medicinal Chemistry, Kunming Institute of Botany, Chinese Academy of Sciences.
E
Fig. 1. Structures of compounds 1–4.
69 70
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O
F
2
The air-dried and powdered whole plants of S. chirayita (5 kg) were extracted with 50% EtOH–H2O (50 L) at room temperature for 2 times, 24 h for each time. The combined EtOH extracts were concentrated in vacuo to yield a brown-yellow gum (1.47 kg), and suspended in H2O (6 L). The suspension was partitioned with petroleum ether (PE) (5 L × 3), EtOAc (5 L × 3) and n-butanol (5 L × 3), successively. The PE part (A, 50 g) was subjected to a silica gel column (7 × 35 cm, 400 g) eluted with H2O–MeOH–CHCl3 (0:0:100, 0:5:95, 1:10:90, 2:20:80, 4:40:60, 5:50:50, v/v, each 1 L). The collected fractions were combined based on their TLC characteristics to yield 7 fractions (Frs. A1– A7). Fr. A2 (3.8 g) was further separated into six subfractions (Frs. A2-1–A2-6), by chromatography over a silica gel column (3 × 33 cm, 100 g) using EtOAc–PE (0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 500 mL each) as the eluent. Fr. A2-4 (1.8 g) was applied to a silica gel column (3 × 28 cm, 80 g, Me2CO–PE, 1:100, 800 mL), to give three subfractions (Frs. A2-4-1–A2-43). Fr. A2-4-3 (293 mg) was purified by a column chromatography (CC) over silica gel (2 × 31 cm, 30 g) with the elution of EtOAc–PE (5:95, 500 mL) yielding 9 (40 mg), 22 (30 mg) and 23 (15 mg). Fr. A2-5 (1.2 g) was subjected to a silica gel CC (2 × 30 cm, 40 g; EtOAc–PE, 5:95–50:50 gradient, 1 L) to afford three fractions (Frs. A2-5-1–A2-5-3). Compound 8 (69 mg) was obtained from Fr. A2-5-3 (260 mg) by a silica gel CC (2 × 32 cm, 30 g) using EtOAc–PE (25:75, 800 mL) as the eluent. Fr. A4 (4 g) was chromatographed on a silica gel column (3 × 40 cm, 100 g; EtOAc–PE, 5:95–50:50 gradient, 2 L), to obtain 5 subfractions (Frs. A4-1–A4-5). Fr. A4-5 (1 g) was subjected to silica gel CC (2 × 29 cm, 35 g) eluted with Me2CO–PE (2:98, 500 mL) to give compounds 10 (600 mg), 24 (200 mg), 25 (80 mg) and 26 (50 mg). Fr. A7 (7.8 g) was subjected to a silica gel CC (4 × 23 cm, 120 g) using EtOAc–PE (5:95–50:50, each 1 L) as the eluent and further purified by a sephadex LH-20 column
Please cite this article as: Zhou N-J, et al, Anti-hepatitis B virus active constituents from Swertia chirayita, Fitoterapia (2014), http:// dx.doi.org/10.1016/j.fitote.2014.11.011
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181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204
F
R O O
179 180
Compounds 2 (2 mg) and 4 (2 mg) was each refluxed with 2 M HCl (5 mL) at 80 °C for 5 h. After neutralization with NaHCO3 and extraction with CH2Cl2, the aqueous layer was concentrated and detected by TLC over silica gel (H2O–MeOH– CHCl3, 4:40:60). The presence of glucose was confirmed by comparison with authentic samples (Rf 0.40). The glucose hydrolyzed from compounds 2 and 4 was purified by Si CC and identified to be D-glucose based on its [α]D value ([α25 D ]: +45.5, c 0.16, H2O; [α25 D ]: +55.7, c 0.14, H2O, respectively) [47].
218
2.5. Anti-HBV assay on HepG 2.2.15 cell line in vitro
227
219 220 221 222 223 224 225 226
The anti-HBV assay was performed according to our previous 228 report [22], with tenofovir (Jiangxi Chenyang Pharmaceutial Co. 229 Ltd, China, purity N 97.6%) as the positive control. 230
2.3.1. Swertiachiralatone A (1) Colorless gum; [α24 D ]: +24.0 (MeOH, c = 0.12,); IR (KBr): νmax = 3417, 1712, 1639, 1476, and 1405 cm−1; 1H NMR and 13 C NMR data, see Table 1; positive HRESIMS: m/z = 223.0921 (calcd for C10H16O4Na, [M + Na]+, −2.0 mDa). 2.3.2. Swertiachoside A (2) Colorless gum; [α24 D ]: −24.9 (MeOH, c = 0.25,); UV (MeOH): λmax (log ε) = 240 (4.8) and 194 (2.5) nm; IR (KBr): νmax = 3423, 1650, 1436, 1411, 1380, 1349, 1316, 1076, and 1039 cm−1; 1H NMR and 13C NMR data, see Table 2; positive HRESIMS: m/z = 415.2343 (calcd for C21H35O8, [M + H]+, 1.7 mDa).
t1:1 t1:2
Table 1 NMR data of swertiachiralatone A (1) in C5D5N (500 Hz for 1H and 125 Hz for 13C).
U
205 206
2.3.3. Swertiachirdiol A (3) White powder, [α24 D ]: +43.9 (CHCl3, c = 0.07); UV (MeOH): λmax (log ε) = 250 (4.0) nm; IR (KBr): νmax =3348,
3. Results and discussion
231
3.1. Structural elucidation
232
Compound 1 colorless gum, had a molecular formula of C10H16O4 based on positive HRESIMS (223.0921 [M + Na]+, calcd. 223.0941) indicating 3° of unsaturation. The IR absorptional bands at 3417, 1712 and 1639 cm− 1 suggested the existence of hydroxyl, carbonyl and olefinic groups. The 1H NMR spectrum (Table 1) displayed signals ascribed to one ethenyl unit [δH 5.90 (1H, m, H-7), 5.17 (2H, m, H-9)], three oxygenated methylenes [δH 4.59 (1H, dd, J = 10.5, 3.0 Hz, H11α) and 4.10 (1H, dd, J = 10.5, 3.0 Hz, H-11β); δH 4.48 (1H, dd, J = 10.8, 1.8 Hz, H-6α), and 4.31 (1H, dd, J = 10.5, 3.1 Hz, H-6β); δH 3.91 (2H, m, H-10)]. Its 13C NMR (DEPT) spectrum (Table 1) revealed the presence of ten carbons including five CH2, four CH and one quaternary carbon, of which one terminal double bond [δC 134.6, (C-7), 118.7 (C-9)] and one carbonyl group [δC 172.9 (C-2)] were characterized. From the above analyses, two of the three degrees of
233 234
t1:3
Position
δC
δH (J in Hz)
Position
δC
δH (J in Hz)
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10
2 3 4 5 6α 6β 7
172.9 (s) 48.9 (d) 33.9 (d) 39.5 (d) 72.6 (t)
– 2.66 (td, 9.3, 2.7) 2.98 (tt, 9.2, 4.6) 2.84 (dd, 7.2, 3.3) 4.48 (dd, 10.8, 1.8) 4.31 (dd, 10.8, 3.1) 5.90 (m)
8α 8β 9 10 11α 11β
34.7 (t) 1.71, tt, 17.2, 8.6
1.72 (td, 13.1, 6.8) 1.91 (td, 13.1, 6.8) 5.17 (m) 3.91 (m) 4.59 (dd, 10.5, 3.0) 4.10 (dd, 10.5, 3.0)
134.6 (d)
215 216 217
P
177 178
213 214
2.4. Acid hydrolysis of compounds 2 and 4
D
175 176
211 212
E
173 174
210
T
171 172
C
169 170
E
167 168
2.3.4. Swertiachoside B (4) Light yellow powder, [α24 D ]: +10.5 (MeOH, c = 0.11); UV (MeOH): λmax (log ε) = 298 (3.6), 259 (4.1), and 207 (4.2) nm; IR (KBr): νmax = 3440, 1653, 1644, 1633, 1627, 1471, 1464, 1456, 1419, 1386, 1375, 1167, 1096, 1070, and 1038 cm−1; 1H NMR and 13C NMR data, see Table 4; positive HRESIMS: m/z = 429.1162 (calcd for C19H25O11, [M + H]+, 3.2 mDa).
R
165 166
R
164
1700, 1653, 1466, 1387, and 1364 cm−1; 1H NMR and 13C NMR 207 data, see Table 3; positive HRESIMS: m/z = 717.5763 (calcd for 208 C46H78O4Na, [M + Na]+, −2.9 mDa). 209
O
162 163
(1.3 × 135 cm, 53 g) eluted with MeOH to give compounds 4 (20 mg), 27 (20 mg) and 28 (30 mg). The EtOAc part (B, 185 g) was chromatographed on a silica gel column (10 × 34 cm, 1000 g) eluted with H2O–MeOH– CHCl3 (0:5:95, 1:10:90, 2:20:80, 4:40:60, 1 L each) to yield six subfractions (Fr. B1–B6). Fr. B2 (1.2 g) was subjected to a silica gel column (2 × 30 cm, 30 g) eluted with MeOH–CHCl3 (1:200, 800 mL) to give compound 11 (20 mg) and Fr. B2-2 (600 mg). Compounds 13 (10 mg), 20 (15 mg), 21 (20 mg) and Fr. B22-2 (200 mg) were obtained from Fr. B2-2 by a silica gel CC (2 × 25 cm, 20 g) using EtOAc–PE (30:70, 500 mL) as the eluent. Fr. B2-2-2 was chromatographed on a silica gel CC (2 × 23 cm, 18 g, Me2CO–PE, 20:80, 500 mL) to yield compound 12 (50 mg). Fr. B3 (1.6 g) was further divided into three subfractions (Frs. B3-1–B3-3), by a silica gel CC (2 × 35 cm, 35 g) using Me2CO–PE (20:80, 1 L) as the eluent. Fr. B3-3 was subjected to a silica gel CC (2 × 30 cm, 30 g) eluted with H2O– CH3OH–CHCl3 (1:10:90, 800 mL) and further purified over a sephadex LH-20 CC (1.3 × 135 cm, 53 g) with an isocratic solvent system of MeOH–CHCl3 (50:50) to yield compounds 5 (70 mg), 17 (5 mg) and 19 (20 mg). Fr. B4 (11 g) was chromatographed on a silica gel column (4 × 39 cm, 200 g) successively eluted with H2O–MeOH–CHCl3 system (0:5:95, 1:10:90, 2:20:80, 4:40:60, each 1 L), to yield four sub-fractions (Fr. B4-1–B4-5). Fr. B4-2 (1 g) was chromatographed on a silica gel CC (2 × 30 cm, 30 g, EtOAc-PE, 10:90, 1.2 L) to yield compounds 6 (7 mg), 14 (15 mg), 15 (30 mg) and 16 (20 mg). Fr. B4-3 (700 mg) was purified by HPLC on a Rp-18 column (9.4 × 250 mm) eluted with MeOH– H2O (30:70, 800 mL) to obtain compounds 2 (20 mg), 18 (15 mg) and 30 (40 mg). Fr. B5 (8 g) was subjected to silica gel CC (4 × 25 cm, 130 g) with the eluent of MeOH–EtOAc (10:90, 1 L) to yield compounds 4 (80 mg), 7 (40 mg) and 29 (40 mg).
N C
160 161
3
118.7 (t) 59.5 (t) 61.7 (t)
Please cite this article as: Zhou N-J, et al, Anti-hepatitis B virus active constituents from Swertia chirayita, Fitoterapia (2014), http:// dx.doi.org/10.1016/j.fitote.2014.11.011
235 236 237 238 239 240 241 242 243 244 245 246 247 248
4
Table 2 NMR data of swertiachoside A (2) in CD3OD (500 Hz for 1H and 100 Hz for 13C).
249 250
202.3 (s) 127.0 (d) 167.9 (s) 49.0 (d) 24.1 (t) 45.7 (d) 23.0 (t) 41.0 (t) 38.7 (s)
2.24 (d, 16.0) 2.15 (d, 16.0) – 5.85 (s) – 2.42 (d, 12.3) 2.15 (overlapped) 1.23 (d, 12.6) 1.81 (tt, 12.0, 2.5) 1.62 (d, 12.2) 1.36 (d, 12.9) 1.53 (d, 12.6) 1.44 (m) –
11 12α 12β 13 14 15 1′ 2′ 3′ 4′ 5′ 6′α 6′β
t3:1 t3:2
Table 3 NMR data of swertiachirdiol A (3) in CDCl3 (400 Hz for 1H and 100 Hz for 13C).
265 266
C
E
263 264
R
261 262
R
259 260
O
257 258
75.1 (s) 76.9 (t) 20.5 (q) 17.0 (q) 22.3 (q) 105.0 (d) 75.2 (d) 77.9 (d) 71.6 (d) 78.0 (d) 62.7 (t)
– 3.97 (d, 10.0) 3.34 (d, 10.0) 1.11 (s) 0.85 (s) 1.96 (s) 4.25 (d, 8.0) 3.20 (m) 3.34 (m) 3.26 (m) 3.29 (m) 3.84 (d, 11.5) 3.63 (d, 11.5)
groups. Its NMR spectral data (Table 2) were similar to those of isopterocarpolone [48], except that one methyl in isopterocarpolone was changed to be O-substituted methylene [δC 76.9 (C-12)] in 2, together with an additional glucosyl moiety [δC 105.0 (C-1′), 75.2 (C-2′), 77.9 (C-3′), 71.6 (C-4′), 78.0 (C-5′), 62.7 (C-6′)]. Hydrolysis of compound 2 yielded Dglucose which was identified by comparing with the authentic sample on TLC and [α]D experiment ([α25 D ]: +45.5, c 0.16, H2O). In the HMBC spectrum, the correlations from H-12 to C-1′ and from H-1′ to C-12 suggested the glycosidation at C-12. The relative stereocenters of C-5, C-7 and C-10 were deduced to be identical with those of isopterocarpolone based on the correlations of H-14 with H-1β, H-5 with H-1α and H-7, and H-8β with H-12 in the ROESY spectrum (Fig. 2). Similar to the previously reported atractyloside I [49], the stereochemistry of C-11 was not determined according to the current experimental data. Up to now, only a few cases of sesquiterpenoids with the 11, 12dihydroxylation pattern were isolated from natural sources, which faced the common problem of the configuration of C-11. Therefore, the structure of 2 was determined as
δH (J in Hz)
Position
25.2 (t) 80.3 (d) 38.1 (s) 55.1 (d) 17.4 (t) 32.6 (t) 43.4 (s)
2.75 (dt, 11.0, 2.8) 1.01 (dt, 11.0, 2.8) 1.63 (overlapped) 4.49 (dd, 11.7, 4.6) – 0.80 (overlapped) 1.42 (overlapped) – –
20 21 22 23 24 25 26 27 28α
9
61.7 (d)
2.35 (s)
28β
10 11 12 13 14 15 16 17 18 19
37.0 (s) 199.9 (s) 128.3 (d) 169.4 (s) 45.5 (s) 25.8 (t) 21.5 (t) 36.9 (s) 42.7 (d) 44.9 (t)
– – 5.57 (s) – – 1.63 (overlapped) – – 2.13 (dd, 10.5, 3.0) 1.72 (d, 13.5)
29 30 1′ 2′ 3′ 4′–13′ 14′ 15′ 16′
t3:3
Position
t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12
1α 1β 2 3 4 5 6 7 8
t3:13 t3:14 t3:15 t3:16 t3:17 t3:18 t3:19 t3:20 t3:21 t3:22 t3:23
δC
C
255 256
38.8 (t)
N
253 254
U
251 252
δH (J in Hz)
T
267 268
unsaturation were deduced, and the leaving one unsaturation requires one ring contained in the structure. A δ-lactone moiety was constructed from the 1H–1H COSY correlations of H-3/H-4/H-5/H-6 and HMBC correlations form H-6 to C-2 and from H-4 to C-2. The 1H–1H COSY correlations of H-5/H-7, H-4/H-8, and H-3/H-11 suggested the direct linkages of C-5 with C-7, C-4 with C-8, and C-3 with C-11. In the ROESY spectrum, the cross-peaks of H-4 with H-5 and H-11, and H-3 and H-7 with H-8 suggested the α-orientation of H-4 and H-5 and β-orientation of H-3 (Fig. 2). Thus, the structure of compound 1 was characterized as (3S, 4S, 5R)-4(2-hydroxyethyl)-3-(hydroxymethyl)-5-vinyltetrahydro2H-pyran-2-one and named as swertiachiralatone A (1). Compound 2 was deduced with the molecular formula C21H34O8 by HRESIMS (m/z 415.2343 [M + H]+, calcd. 415.2326). The IR spectrum showed the presence of hydroxyl (3423 cm− 1), double bond (1650 cm− 1) and glycosidic linkage (1076 cm− 1) in the structure. The 13C NMR (DEPT) spectrum exhibited 21 carbons, including 4 quaternary carbons, 9 methines, 5 methylenes and 3 methyl
δC
F
55.2 (d)
Position
O
1α 1β 2 3 4 5 6α 6β 7 8α 8β 9α 9β 10
δH (J in Hz)
R O
t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17
δC
P
Position
D
t2:3
E
t2:1 t2:2
N.-J. Zhou et al. / Fitoterapia xxx (2014) xxx–xxx
δC
δH (J in Hz) 31.1 (s) 31.9 (t) 30.7 (t) 28.1 (q) 16.4 (q) 16.8 (q) 18.6 (q) 23.4 (q) 69.7 (t)
33.0 (q) 23.4 (q) 173.7 (s) 34.9 (t) 33.8 (t) 29.7–29.2 (t) 23.6 (t) 22.7 (t) 14.2 (q)
– – 1.42 (overlapped) 0.87 (overlapped) 1.15 (s) 0.87 (overlapped) 1.10 (s) 1.38 (s) 3.45 (d, 10.9) 3.22, d, 12.0 3.19 (d, 10.9) 3.22, d, 12.0 0.92 (s) 0.87 (overlapped) – 2.27 (t, 7.5) – – – 1.94 (m) 0.87 (overlapped)
Please cite this article as: Zhou N-J, et al, Anti-hepatitis B virus active constituents from Swertia chirayita, Fitoterapia (2014), http:// dx.doi.org/10.1016/j.fitote.2014.11.011
269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288
N.-J. Zhou et al. / Fitoterapia xxx (2014) xxx–xxx t4:1 t4:2
5
Table 4 NMR data of swertiachoside B (4) in CD3OD (400 Hz for 1H and 400 Hz for 13C). Position
δC
δH (J in Hz)
Position
δC
δH (J in Hz)
t4:4 t4:5 t4:6 t4:7 t4:8 t4:9 t4:10 t4:11 t4:12 t4:13 t4:14
1 2 3 4 4a 4b 5 6 7α 7β 8
163.4 (s) 100.2 (d) 166.2 (s) 95.1 (d) 159.3 (s) 168.4 (s) 67.6 (d) 27.4 (t) 27.9 (t)
– 6.17 (d, 2.0) – 6.35 (d, 2.0) – – 4.58 (dd, 9.5, 7.0) 2.06 (m) 2.29 (dq, 13.3, 3.0) 1.76 (t, 13.3) 4.94 (overlapped)
8a 8b 9 1′ 2′ 3′ 4′ 5′ 6′α 6′β
117.8 (s) 105.3 (s) 183.0 (s) 105.2 (d) 71.7 (d) 78.0 (d) 75.8 (d) 78.2 (d) 62.9 (t)
– – – 4.68 (d, 7.6) 3.19 (dd, 12.0, 2.0) – – – 3.88 (dd, 12.0, 2.0) 3.69 (dd, 12.0, 2.0)
R O O
P
D E T C E R R
295
O
293 294
methyl groups and one axial proton [δH = 4.49 (1H, dd, J = 11.7, 4.6 Hz, H-3)]. The13C NMR (DEPT) data showed 46 carbon signals ascribed to 8 methyls [δC 28.1 (C-23), 16.4 (C-24), 16.8 (C-25), 18.6 (C-26), 23.4 (C-27), 33.0 (C-29), 23.4 (C-30), 14.2 (C-16′)], 24 methylenes, 5 methines and 9 quaternary carbon atoms, from which one olean-type triterpene motif and one long-chain fatty acid moiety were easily recognized. The 1H
N C
291 292
isopterocarpolone 12-O-β-D-glucopyranoside and named as swertiachoside A (2). Compound 3 had a molecular formula of C46H78O4 from HRESIMS at m/z 717.5763 [M + Na]+ (calcd for 717.5792). Its IR spectrum showed absorptional bands at 3448 and 1653 cm− 1 ascribed to hydroxyl and carbonyl functionalities. Its 1H NMR spectrum (Table 3) displayed signals of several
U
289 290
71.3 (d)
F
t4:3
Fig. 2. Key 2D NMR correlations of compounds 1–4.
Please cite this article as: Zhou N-J, et al, Anti-hepatitis B virus active constituents from Swertia chirayita, Fitoterapia (2014), http:// dx.doi.org/10.1016/j.fitote.2014.11.011
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N.-J. Zhou et al. / Fitoterapia xxx (2014) xxx–xxx
314 315 316 317 318 319 320 321 322 323 324 325
t5:3
Compounds
HBsAgb
CC50 [mM]
b c d e f
0.18
0.043 0.36 0.44
0.076
e
C
SI
– – – – – – – – – b1 – – – 4.29 – – 0.20 – 1.23 – – – – – – – – – – – N1.39
E
R
R
0.55
N4.75 N2.04 N1.88 N2.49 N4.14 N2.15 N3.03 N1.81 N2.56 0.40 ± N0.88 N4.53 N1.98 0.31 ± N3.41 N3.79 1.48 ± N3.04 1.49 ± N3.20 N3.28 N10.49 N1.67 N0.65 N1.19 N1.00 N3.22 N7.18 N1.45 N2.68 1.25 ±
O
0.21 0.0038
C
a
0.55 0.12
N
t5:35 t5:36 t5:37 t5:38 t5:39 t5:40
N4.75 N2.04 N1.88 N2.49 3.43 ± 1.34 ± N3.03 N1.81 1.38 ± 0.02 ± N0.88 4.25 ± N1.98 1.35 ± N3.41 N3.79 0.30 ± N3.04 1.83 ± 2.73 ± N3.28 N10.49 N1.67 N0.65 N1.19 N1.00 N3.22 N7.18 0.66 ± N2.68 N1.74
U
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Tenofovirf
342
Compounds 1–30 was evaluated for their anti-HBV activity on HepG 2.2.15 cells in vitro, namely inhibiting the secretions of HBsAg and HBeAg, and HBV DNA replication using tenofovir as the positive control [22]. As shown in Table 5, compounds 10, 14, 17, 19 showed activity inhibiting HBsAg secretion; compounds 14 and 28 showed activity inhibiting HBeAg
343
HBeAgc
IC50 [mM] t5:4 t5:5 t5:6 t5:7 t5:8 t5:9 t5:10 t5:11 t5:12 t5:13 t5:14 t5:15 t5:16 t5:17 t5:18 t5:19 t5:20 t5:21 t5:22 t5:23 t5:24 t5:25 t5:26 t5:27 t5:28 t5:29 t5:30 t5:31 t5:32 t5:33 t5:34
3.2. In vitro anti-HBV activity
F
312 313
O
310 311
T
308 309
R O
Table 5 Anti-HBV activities of compounds 1–30a.
307
327 328
P
t5:1 t5:2
305 306
data (Table 2) were similar to those of tetrahydroswertianolin [50], except for the absence of the methoxy group at C-3, from which compound 4 was proposed to be the 3-O-demethylated derivative of tetrahydroswertianolin. The full NMR data assignments of compound 4 were performed with the aid of the HSQC, 1H–1H COSY and HMBC data (Fig. 2). In the HMBC spectrum, the correlation of H-1′ with C-8 indicated that the glucosyl group was attached to C-8. The cross-peaks of H-5 with H-8 in the ROESY spectrum suggested that H-5 and H-8 were located at the same side. Its absolute configuration was determined to be identical with tetrahydroswertianolin by their similar [α]D values (tetrahydroswertianolin: [α 24 D ]: +8.0, c 0.2, MeOH; compound 4: [α 24 D ]: +10.5, c 0.11, MeOH). Hence, compound 4 was defined to be 3-nortetrahydroswertianolin and named as swertiachoside B (4) as shown in Fig. 1.
E
326
and 13C NMR (DEPT) data of compound 3 were almost identical to those of 3β-hydroxy-11-oxo-olean-12-enyl-3-palmitate (24) [40], except that one methyl (C-28) in 24 was changed to hydroxymethyl group [δC 69.7 (C-28)] in 3, which was supported by the HMBC correlations of H-28 with C-16, C-17, C-18 and C-22 (Fig. 2). The palmityloxy group was proposed at C-3 position from the HMBC correlation of H-3 to C-1′. The H-3 was determined to be an α-orientation from the splitting and coupling constants (J) of the proton signal at 4.49 (dd, J = 11.7, 4.6 Hz), and the ROESY correlation of H-3 with H-23. Similarly, the ROESY correlations of H-25 with H-24 and H-26, H-9 with H-5 and H-27 indicated the α-orientation of H-5, H-9 and Me-27 and the β-orientation of Me-24, Me-25 and Me-26. Consequently, the structure of compound 3 was determined as 3β, 28-dihydroxy-11-oxo-olean-12-enyl-3-palmitate and named as swertiachirdiol A (3). Compound 4 possessed a molecular formula of C19H22O11 determined by HRESIMS at m/z 425.1081 [M – H]+ (calcd for 425.1084). The IR spectrum suggested the presence of hydroxyl (3440 cm−1) and hydrogen bonded ketone (1653 cm−1) groups. Hydrolysis of compound 4 with 2 M HCl in methanol to yield D-glucose which was identified by comparing with the authentic sample on TLC, as well as the [α]D experiment 1 13 ([α 25 C NMR (DEPT) spectral D ]: +55.7, c 0.14, H2O). Its H and
303 304
D
6
0.035
0.045
0.23 0.33
0.025
DNAd
IC50 [mM]
SI
IC50 [mM]
SI
N4.75 N2.04 N1.88 N2.49 N4.14 N2.15 N3.03 N1.81 N2.56 N1.47 N0.88 N4.53 N1.98 0.77 ± 0.076 N3.41 N3.79 N2.25 N3.04 N2.43 N3.20 N3.28 N10.49 N1.67 N0.65 N1.19 N1.00 N3.22 5.92 ± 1.02 N1.45 N2.68 1.21 ± 0.031
– – – – – – – – – – – – – 1.75 – – – – – – – – – – – – – N1.21 – – N1.44
N1.19 N0.51 N0.47 N0.62 N1.03 N0.54 N0.76 0.30 ± 0.051 0.070 ± 0.010 N0.47 N0.22 N1.13 0.16 ± 0.021 0.29 ± 0.034 N0.85 N0.95 0.33 ± 0.055 N0.76 N0.61 N0.80 N0.82 N2.62 N0.42 0.22 ± 0.045 0.13 ± 0.021 0.24 ± 0.045 N0.80 N1.80 0.22 ± 0.037 N0.67 0.0011 ± 0.00043
– – – – – – – N6.03 19.71 – – – N12.38 4.66 – – b1 – – – – – – N4.57 N5.07 N4.85 – – 2.97 – N1581.82
All values are the mean of two independent experiments. HBsAg: HBV surface antigen. HBeAg: HBV e antigen. DNA: HBV DNA replication. CC50 = 50% cytotoxic concentration, IC50 = 50% inhibition concentration, SI (selectivity index) = CC50 / IC50. Tenofovir, an antiviral agent used as a positive control.
Please cite this article as: Zhou N-J, et al, Anti-hepatitis B virus active constituents from Swertia chirayita, Fitoterapia (2014), http:// dx.doi.org/10.1016/j.fitote.2014.11.011
329 330 331 332 333 334 335 336 337 338 339 340 341
344 345 346 347 348
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369
Four new compounds, involving one unusual eudesmane sesquiterpene and one tetrahydroxanthone, together with twenty-six known compounds were firstly isolated from S. chirayita. Eleven compounds showed anti-HBV activities on HepG 2.2.15 cells line in vitro (four compounds showed activity inhibiting HBsAg secretion, two compounds showed activity inhibiting HBeAg secretion, and nine compounds exhibited activity inhibiting HBV DNA replication). This work will provide valuable information for revealing the anti-HBV active constituents of S. chirayita.
370
5. Statement
361 362 363 364 365 366 367 368
371
The authors declare no conflict of interest.
T
356 357
Acknowledgments
373 374
379
This work was supported by the International Foundation for Science in Sweden (No. F/5202-1), the National Natural Science Foundation of China for Distinguished Young Scholars (No. 81025023), the West Light Foundation of the Chinese Academy of Sciences, the National Natural Science Foundation of China (No. 81202436), and the Youth Innovation Promotion Association, CAS.
380
Appendix A. Supporting information
381
384
1D and 2D NMR, HREIMS/HRESIMS, IR, UV, [α]D spectra of compounds 1–4 are available as Supporting information. Supplementary data related to this article can be found online at doi: http://dx.doi.org/10.1016/j.fitote.2014.11.011
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secretion; and compounds 8, 9, 13, 14, 17, 24–26 and 29 exhibited activity inhibiting HBV DNA replication. In particular, compound 13 displayed obviously inhibition on HBV DNA replication with IC50 value of 0.16 ± 0.021 mM, with high SI value of N12.38. (+)-Cycloolivil-4′-O-β-D-glucopyranoside (14) exhibited inhibition not only on the secretions of HBsAg and HBeAg with IC50 values of 0.31 ± 0.045 mM (SI = 4.29) and 0.77 ± 0.076 mM (SI = 1.75), respectively, but also on HBV DNA replication with an IC50 value of 0.29 ± 0.034 mM (SI = 4.66).
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Contents lists available at ScienceDirect
Fitoterapia
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Anti-hepatitis B virus active constituents from Swertia chirayita
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journal homepage: www.elsevier.com/locate/fitote
Ning-Jia Zhou a,c, Chang-An Geng a, Xiao-Yan Huang a, Yun-Bao Ma a, Xue-Mei Zhang a, Ju-Le Wang b, Ji-Jun Chen a,⁎ a b c
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China Medical School of Tibet University, Lhasa 850000, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China
9
a r t i c l e
i n f o
10 14 11 12 13
Article history: Received 2 September 2014 Accepted in revised form 10 November 2014 Available online xxxx
28 29 Q2 30 31 32 33 34
Keywords: Swertiachiralatone A Swertiachoside A Swertiachirdiol A Swertiachoside B Anti-hepatitis B virus activity Swertia chirayita
35
1. Introduction
36 37
Swertia chirayita belonging to the family Gentianaceae is distributed throughout the temperate Himalaya area (Kashmir to Bhutan, and Kashia hills) between 1200 and 3000 m above sea level. The whole plant is widely used by local people for the treatment of hepatitis, inflammation and digestive diseases [1]. Previous studies on this species led to the isolation of twenty-one xanthones, eleven triterpenoids, five secoiridoids, as well as eight other compounds including alkaloids, phenolic compounds and steroids [2–18]. Our previous investigation on Swertia mileensis, the congener plant of S. chirayita, resulted in a series of novel lactones with unprecedented structures and promising antihepatitis B virus (anti-HBV) activity [19–22]. According to our preliminary bioassay in vitro, the 50% EtOH–H2O extract of S. chirayita could inhibit the secretions of hepatitis B surface
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a b s t r a c t
41 42 43 44Q3 45 46 47 48 49
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Four new compounds swertiachiralatone A (1), swertiachoside A (2), swertiachirdiol A (3) and swertiachoside B (4), together with twenty-six known ones were isolated from the ethanol extract of Swertia chirayita. Their structures were elucidated by extensive spectroscopic analyses (1D- and 2D-NMR, HRESIMS, UV, IR and [α]D). All compounds were evaluated for anti-hepatitis B virus (anti-HBV) activities on HepG 2.2.15 cells line in vitro, of which compounds 14 and 19 showed inhibitory activity on hepatitis B surface antigen (HBsAg) secretion with IC50 values of 0.31 ± 0.045 and 1.49 ± 0.033 mM; compounds 14 and 28 exhibited activity against hepatitis B e antigen (HBeAg) secretion with IC50 values of 0.77 ± 0.076 and 5.92 ± 1.02 mM; and eight compounds (8,9,13,14,24–26,29) possessed activity against HBV DNA replication with IC50 values of 0.07–0.33 mM. In particular (+)-cycloolivil-4′-O-β-D-glucopyranoside (14) exhibited inhibition not only on the secretions of HBsAg and HBeAg with IC50 values of 0.31 ± 0.045 mM (SI = 4.29) and 0.77 ± 0.076 mM (SI = 1.75), respectively, but also on HBV DNA replication with an IC50 value of 0.29 ± 0.034 mM (SI = 4.66). © 2014 Published by Elsevier B.V.
⁎ Corresponding author. Tel.: +86 871 65223265; fax: +86 871 65227197. E-mail address: [email protected] (J.-J. Chen).
antigen (HBsAg) and hepatitis B e antigen (HBeAg) with IC50 values of 1.08 ± 0.23 mg/mL (SI N 2.0) and 0.11 ± 0.031 mg/mL (SI N 19.6), respectively, as well as HBV DNA replication with an IC50 value of 0.17 ± 0.025 mg/mL (SI = 12.9). However, the components responsible for anti-HBV activity in S. chirayita were still unclear. In order to clarify the active constituents, bioassay-guided fractionation of the 50% EtOH–H2O extract resulted in four new compounds (Fig. 1), swertiachiralatone A (1), swertiachoside A (2), swertiachirdiol A (3) and swertiachoside B (4), together with twenty-six known ones, isoorientin (5) [23], 2-C-β-D-glucopy-ranosyl-1, 3, 7trihydroxyxanthane (6) [24], mangiferin (7) [25], 8-O-[β-Dxylopyranosyl-(1 → 6)-β-D-glucopyranosyl]-1, 7-dihydroxyl3-methoxyxanthone (8) [26], 8-O-[β-D-xylopyranosyl-(1 → 6)β-D-glucopyranosyl]-1-hydroxyl-3, 7-dimethoxy-xanthone (9) [27], 1-O-β-D-glucopyranosyl-3, 5, 8-trihydroxyxanthone (10) [28], 7-O-[β-D-xylopyranosyl-(1 → 2)-β-D-xylopyranosyl]1, 8-dihydroxy-3-methoxyxanthone (11) [29], 1, 5, 8trihydroxyl-3-methoxyxanthone (12) [30], 1-hydroxy-3, 7-
http://dx.doi.org/10.1016/j.fitote.2014.11.011 0367-326X/© 2014 Published by Elsevier B.V.
Please cite this article as: Zhou N-J, et al, Anti-hepatitis B virus active constituents from Swertia chirayita, Fitoterapia (2014), http:// dx.doi.org/10.1016/j.fitote.2014.11.011
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N.-J. Zhou et al. / Fitoterapia xxx (2014) xxx–xxx
spectra were taken on a Shimadzu UV-2401PC spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were measured on a Bio-Rad FTS-135 spectrometer (Bio-Rad, Hercules, California, USA) with KBr pellets. 1D and 2D-NMR spectra were recorded on Bruker AM-400 or DRX-500 spectrometers (Bruker, Bremerhaven, Germany). Silica gel (200–300 mesh) for column chromatography was obtained from Qingdao Makall Chemical Company (Makall, Qingdao, China). Semi-preparative HPLC was conducted on Waters Alliance 2695 liquid chromatography with a ZORBAX SB-C18 (5 μm, 9.4 × 250 mm) column (Agilent, USA). Sephadex LH-20 (20–150 μm, Pharmacia, Sweden) and ODS (45–70 μm, Merck, Germany) were used for column chromatography. Fractions were monitored by thin-layer chromatography (TLC), and spots were visualized by heating silica gel plates after sprayed with 10% H2SO4 in EtOH.
104 105
2.2. Plant material
119
P
2. Experimental
100
2.1. General experimental procedures
101 102
Mass spectra were run on LCMS-IT-TOF spectrometer (Shimadzu, Kyoto, Japan). Optical rotations were obtained on a JASCO model 1020 polarimeter (Horiba, Tokyo, Japan). UV
84 85 86 87 88 89 90 91 92 93 94 95 96 97
103
C
E
R
R
83
O
81 82
C
79 80
N
77 78
U
75 76
D
99
73 74
2.3. Extraction and isolation
T
98
dimethoxyxanthone (13) [31], (+)-cycloolivil-4′-O-β-Dglucopyranoside (14) [32], 8′-α-hydroxyl-lariciresinol-4-Oβ-D-glucopyranoside (15) [33], 8′-α-hydroxyllariciresinol4′-O-β-D-glucopyranoside (16) [34], 3, 4′-dihydroxy-3′methoxypropiophenone 3-O-β-D-glucopyranoside (17) [35], epi-syringaresinol-4″-O-β-D-glucopyranoside (18) [36], syringaresinol 4″-O-β-D-glucopyranoside (19) [37], 6′-Oβ-D-glucopyranosylgentiopicroside (20) [38], 6′-O-β-Dglucopyranosylsweroside (21), djalonenol (22) [39], swerbimalactone B (23), 3β-hydroxy-11-oxo-olean-12-enyl3-palmitate (24) [40], erythrodiol-3-O-palmitate (25) [41], olean-12-ene-28-carboxy-3β-hexadecanoate (26) [42], cholest4-en-3-one (27) [43], 3, 3′, 5-trihydroxybiphenyl (28) [44], bridelionoside B (29) [45], and (6R, 7E, 9R)-9-hydroxymegasigma-4, 7-dien-3-one-9-O-β-D-glucopyranoside (30) [46]. All the known compounds were isolated from this plant for the first time. Isolates 1–30 were evaluated for their antiHBV activities on HepG 2.2.15 cells line in vitro, of which compounds 14 and 19 showed inhibitory activity on HBsAg secretion with IC50 values of 0.31 ± 0.045 and 1.49 ± 0.033 mM; compounds 14 and 28 showed activity against HBeAg secretion with IC50 values of 0.77 ± 0.076 and 5.92 ± 1.02 mM; and eight compounds (8,9,13,14,24–26,29) exhibited activity against HBV DNA replication with IC50 values of 0.07–0.33 mM. In particular (+)-cycloolivil-4′-O-β-Dglucopyranoside (14) exhibited inhibition not only on the secretions of HBsAg and HBeAg with IC50 values of 0.31 ± 0.045 mM (SI = 4.29) and 0.77 ± 0.076 mM (SI = 1.75), respectively, but also on HBV DNA replication with an IC50 value of 0.29 ± 0.034 mM (SI = 4.66).
71 72
The whole plant of Swertia chirayita (Roxb. ex Fleming) H. Karst. was collected in Nepal, in September 2008 and identified by Prof. Ju-Le Wang, Medical School of Tibet University. A voucher specimen (No. 2008-11-30) was deposited at the Laboratory of Antivirus and Natural Medicinal Chemistry, Kunming Institute of Botany, Chinese Academy of Sciences.
E
Fig. 1. Structures of compounds 1–4.
69 70
R O
O
F
2
The air-dried and powdered whole plants of S. chirayita (5 kg) were extracted with 50% EtOH–H2O (50 L) at room temperature for 2 times, 24 h for each time. The combined EtOH extracts were concentrated in vacuo to yield a brown-yellow gum (1.47 kg), and suspended in H2O (6 L). The suspension was partitioned with petroleum ether (PE) (5 L × 3), EtOAc (5 L × 3) and n-butanol (5 L × 3), successively. The PE part (A, 50 g) was subjected to a silica gel column (7 × 35 cm, 400 g) eluted with H2O–MeOH–CHCl3 (0:0:100, 0:5:95, 1:10:90, 2:20:80, 4:40:60, 5:50:50, v/v, each 1 L). The collected fractions were combined based on their TLC characteristics to yield 7 fractions (Frs. A1– A7). Fr. A2 (3.8 g) was further separated into six subfractions (Frs. A2-1–A2-6), by chromatography over a silica gel column (3 × 33 cm, 100 g) using EtOAc–PE (0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 500 mL each) as the eluent. Fr. A2-4 (1.8 g) was applied to a silica gel column (3 × 28 cm, 80 g, Me2CO–PE, 1:100, 800 mL), to give three subfractions (Frs. A2-4-1–A2-43). Fr. A2-4-3 (293 mg) was purified by a column chromatography (CC) over silica gel (2 × 31 cm, 30 g) with the elution of EtOAc–PE (5:95, 500 mL) yielding 9 (40 mg), 22 (30 mg) and 23 (15 mg). Fr. A2-5 (1.2 g) was subjected to a silica gel CC (2 × 30 cm, 40 g; EtOAc–PE, 5:95–50:50 gradient, 1 L) to afford three fractions (Frs. A2-5-1–A2-5-3). Compound 8 (69 mg) was obtained from Fr. A2-5-3 (260 mg) by a silica gel CC (2 × 32 cm, 30 g) using EtOAc–PE (25:75, 800 mL) as the eluent. Fr. A4 (4 g) was chromatographed on a silica gel column (3 × 40 cm, 100 g; EtOAc–PE, 5:95–50:50 gradient, 2 L), to obtain 5 subfractions (Frs. A4-1–A4-5). Fr. A4-5 (1 g) was subjected to silica gel CC (2 × 29 cm, 35 g) eluted with Me2CO–PE (2:98, 500 mL) to give compounds 10 (600 mg), 24 (200 mg), 25 (80 mg) and 26 (50 mg). Fr. A7 (7.8 g) was subjected to a silica gel CC (4 × 23 cm, 120 g) using EtOAc–PE (5:95–50:50, each 1 L) as the eluent and further purified by a sephadex LH-20 column
Please cite this article as: Zhou N-J, et al, Anti-hepatitis B virus active constituents from Swertia chirayita, Fitoterapia (2014), http:// dx.doi.org/10.1016/j.fitote.2014.11.011
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N.-J. Zhou et al. / Fitoterapia xxx (2014) xxx–xxx
181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204
F
R O O
179 180
Compounds 2 (2 mg) and 4 (2 mg) was each refluxed with 2 M HCl (5 mL) at 80 °C for 5 h. After neutralization with NaHCO3 and extraction with CH2Cl2, the aqueous layer was concentrated and detected by TLC over silica gel (H2O–MeOH– CHCl3, 4:40:60). The presence of glucose was confirmed by comparison with authentic samples (Rf 0.40). The glucose hydrolyzed from compounds 2 and 4 was purified by Si CC and identified to be D-glucose based on its [α]D value ([α25 D ]: +45.5, c 0.16, H2O; [α25 D ]: +55.7, c 0.14, H2O, respectively) [47].
218
2.5. Anti-HBV assay on HepG 2.2.15 cell line in vitro
227
219 220 221 222 223 224 225 226
The anti-HBV assay was performed according to our previous 228 report [22], with tenofovir (Jiangxi Chenyang Pharmaceutial Co. 229 Ltd, China, purity N 97.6%) as the positive control. 230
2.3.1. Swertiachiralatone A (1) Colorless gum; [α24 D ]: +24.0 (MeOH, c = 0.12,); IR (KBr): νmax = 3417, 1712, 1639, 1476, and 1405 cm−1; 1H NMR and 13 C NMR data, see Table 1; positive HRESIMS: m/z = 223.0921 (calcd for C10H16O4Na, [M + Na]+, −2.0 mDa). 2.3.2. Swertiachoside A (2) Colorless gum; [α24 D ]: −24.9 (MeOH, c = 0.25,); UV (MeOH): λmax (log ε) = 240 (4.8) and 194 (2.5) nm; IR (KBr): νmax = 3423, 1650, 1436, 1411, 1380, 1349, 1316, 1076, and 1039 cm−1; 1H NMR and 13C NMR data, see Table 2; positive HRESIMS: m/z = 415.2343 (calcd for C21H35O8, [M + H]+, 1.7 mDa).
t1:1 t1:2
Table 1 NMR data of swertiachiralatone A (1) in C5D5N (500 Hz for 1H and 125 Hz for 13C).
U
205 206
2.3.3. Swertiachirdiol A (3) White powder, [α24 D ]: +43.9 (CHCl3, c = 0.07); UV (MeOH): λmax (log ε) = 250 (4.0) nm; IR (KBr): νmax =3348,
3. Results and discussion
231
3.1. Structural elucidation
232
Compound 1 colorless gum, had a molecular formula of C10H16O4 based on positive HRESIMS (223.0921 [M + Na]+, calcd. 223.0941) indicating 3° of unsaturation. The IR absorptional bands at 3417, 1712 and 1639 cm− 1 suggested the existence of hydroxyl, carbonyl and olefinic groups. The 1H NMR spectrum (Table 1) displayed signals ascribed to one ethenyl unit [δH 5.90 (1H, m, H-7), 5.17 (2H, m, H-9)], three oxygenated methylenes [δH 4.59 (1H, dd, J = 10.5, 3.0 Hz, H11α) and 4.10 (1H, dd, J = 10.5, 3.0 Hz, H-11β); δH 4.48 (1H, dd, J = 10.8, 1.8 Hz, H-6α), and 4.31 (1H, dd, J = 10.5, 3.1 Hz, H-6β); δH 3.91 (2H, m, H-10)]. Its 13C NMR (DEPT) spectrum (Table 1) revealed the presence of ten carbons including five CH2, four CH and one quaternary carbon, of which one terminal double bond [δC 134.6, (C-7), 118.7 (C-9)] and one carbonyl group [δC 172.9 (C-2)] were characterized. From the above analyses, two of the three degrees of
233 234
t1:3
Position
δC
δH (J in Hz)
Position
δC
δH (J in Hz)
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10
2 3 4 5 6α 6β 7
172.9 (s) 48.9 (d) 33.9 (d) 39.5 (d) 72.6 (t)
– 2.66 (td, 9.3, 2.7) 2.98 (tt, 9.2, 4.6) 2.84 (dd, 7.2, 3.3) 4.48 (dd, 10.8, 1.8) 4.31 (dd, 10.8, 3.1) 5.90 (m)
8α 8β 9 10 11α 11β
34.7 (t) 1.71, tt, 17.2, 8.6
1.72 (td, 13.1, 6.8) 1.91 (td, 13.1, 6.8) 5.17 (m) 3.91 (m) 4.59 (dd, 10.5, 3.0) 4.10 (dd, 10.5, 3.0)
134.6 (d)
215 216 217
P
177 178
213 214
2.4. Acid hydrolysis of compounds 2 and 4
D
175 176
211 212
E
173 174
210
T
171 172
C
169 170
E
167 168
2.3.4. Swertiachoside B (4) Light yellow powder, [α24 D ]: +10.5 (MeOH, c = 0.11); UV (MeOH): λmax (log ε) = 298 (3.6), 259 (4.1), and 207 (4.2) nm; IR (KBr): νmax = 3440, 1653, 1644, 1633, 1627, 1471, 1464, 1456, 1419, 1386, 1375, 1167, 1096, 1070, and 1038 cm−1; 1H NMR and 13C NMR data, see Table 4; positive HRESIMS: m/z = 429.1162 (calcd for C19H25O11, [M + H]+, 3.2 mDa).
R
165 166
R
164
1700, 1653, 1466, 1387, and 1364 cm−1; 1H NMR and 13C NMR 207 data, see Table 3; positive HRESIMS: m/z = 717.5763 (calcd for 208 C46H78O4Na, [M + Na]+, −2.9 mDa). 209
O
162 163
(1.3 × 135 cm, 53 g) eluted with MeOH to give compounds 4 (20 mg), 27 (20 mg) and 28 (30 mg). The EtOAc part (B, 185 g) was chromatographed on a silica gel column (10 × 34 cm, 1000 g) eluted with H2O–MeOH– CHCl3 (0:5:95, 1:10:90, 2:20:80, 4:40:60, 1 L each) to yield six subfractions (Fr. B1–B6). Fr. B2 (1.2 g) was subjected to a silica gel column (2 × 30 cm, 30 g) eluted with MeOH–CHCl3 (1:200, 800 mL) to give compound 11 (20 mg) and Fr. B2-2 (600 mg). Compounds 13 (10 mg), 20 (15 mg), 21 (20 mg) and Fr. B22-2 (200 mg) were obtained from Fr. B2-2 by a silica gel CC (2 × 25 cm, 20 g) using EtOAc–PE (30:70, 500 mL) as the eluent. Fr. B2-2-2 was chromatographed on a silica gel CC (2 × 23 cm, 18 g, Me2CO–PE, 20:80, 500 mL) to yield compound 12 (50 mg). Fr. B3 (1.6 g) was further divided into three subfractions (Frs. B3-1–B3-3), by a silica gel CC (2 × 35 cm, 35 g) using Me2CO–PE (20:80, 1 L) as the eluent. Fr. B3-3 was subjected to a silica gel CC (2 × 30 cm, 30 g) eluted with H2O– CH3OH–CHCl3 (1:10:90, 800 mL) and further purified over a sephadex LH-20 CC (1.3 × 135 cm, 53 g) with an isocratic solvent system of MeOH–CHCl3 (50:50) to yield compounds 5 (70 mg), 17 (5 mg) and 19 (20 mg). Fr. B4 (11 g) was chromatographed on a silica gel column (4 × 39 cm, 200 g) successively eluted with H2O–MeOH–CHCl3 system (0:5:95, 1:10:90, 2:20:80, 4:40:60, each 1 L), to yield four sub-fractions (Fr. B4-1–B4-5). Fr. B4-2 (1 g) was chromatographed on a silica gel CC (2 × 30 cm, 30 g, EtOAc-PE, 10:90, 1.2 L) to yield compounds 6 (7 mg), 14 (15 mg), 15 (30 mg) and 16 (20 mg). Fr. B4-3 (700 mg) was purified by HPLC on a Rp-18 column (9.4 × 250 mm) eluted with MeOH– H2O (30:70, 800 mL) to obtain compounds 2 (20 mg), 18 (15 mg) and 30 (40 mg). Fr. B5 (8 g) was subjected to silica gel CC (4 × 25 cm, 130 g) with the eluent of MeOH–EtOAc (10:90, 1 L) to yield compounds 4 (80 mg), 7 (40 mg) and 29 (40 mg).
N C
160 161
3
118.7 (t) 59.5 (t) 61.7 (t)
Please cite this article as: Zhou N-J, et al, Anti-hepatitis B virus active constituents from Swertia chirayita, Fitoterapia (2014), http:// dx.doi.org/10.1016/j.fitote.2014.11.011
235 236 237 238 239 240 241 242 243 244 245 246 247 248
4
Table 2 NMR data of swertiachoside A (2) in CD3OD (500 Hz for 1H and 100 Hz for 13C).
249 250
202.3 (s) 127.0 (d) 167.9 (s) 49.0 (d) 24.1 (t) 45.7 (d) 23.0 (t) 41.0 (t) 38.7 (s)
2.24 (d, 16.0) 2.15 (d, 16.0) – 5.85 (s) – 2.42 (d, 12.3) 2.15 (overlapped) 1.23 (d, 12.6) 1.81 (tt, 12.0, 2.5) 1.62 (d, 12.2) 1.36 (d, 12.9) 1.53 (d, 12.6) 1.44 (m) –
11 12α 12β 13 14 15 1′ 2′ 3′ 4′ 5′ 6′α 6′β
t3:1 t3:2
Table 3 NMR data of swertiachirdiol A (3) in CDCl3 (400 Hz for 1H and 100 Hz for 13C).
265 266
C
E
263 264
R
261 262
R
259 260
O
257 258
75.1 (s) 76.9 (t) 20.5 (q) 17.0 (q) 22.3 (q) 105.0 (d) 75.2 (d) 77.9 (d) 71.6 (d) 78.0 (d) 62.7 (t)
– 3.97 (d, 10.0) 3.34 (d, 10.0) 1.11 (s) 0.85 (s) 1.96 (s) 4.25 (d, 8.0) 3.20 (m) 3.34 (m) 3.26 (m) 3.29 (m) 3.84 (d, 11.5) 3.63 (d, 11.5)
groups. Its NMR spectral data (Table 2) were similar to those of isopterocarpolone [48], except that one methyl in isopterocarpolone was changed to be O-substituted methylene [δC 76.9 (C-12)] in 2, together with an additional glucosyl moiety [δC 105.0 (C-1′), 75.2 (C-2′), 77.9 (C-3′), 71.6 (C-4′), 78.0 (C-5′), 62.7 (C-6′)]. Hydrolysis of compound 2 yielded Dglucose which was identified by comparing with the authentic sample on TLC and [α]D experiment ([α25 D ]: +45.5, c 0.16, H2O). In the HMBC spectrum, the correlations from H-12 to C-1′ and from H-1′ to C-12 suggested the glycosidation at C-12. The relative stereocenters of C-5, C-7 and C-10 were deduced to be identical with those of isopterocarpolone based on the correlations of H-14 with H-1β, H-5 with H-1α and H-7, and H-8β with H-12 in the ROESY spectrum (Fig. 2). Similar to the previously reported atractyloside I [49], the stereochemistry of C-11 was not determined according to the current experimental data. Up to now, only a few cases of sesquiterpenoids with the 11, 12dihydroxylation pattern were isolated from natural sources, which faced the common problem of the configuration of C-11. Therefore, the structure of 2 was determined as
δH (J in Hz)
Position
25.2 (t) 80.3 (d) 38.1 (s) 55.1 (d) 17.4 (t) 32.6 (t) 43.4 (s)
2.75 (dt, 11.0, 2.8) 1.01 (dt, 11.0, 2.8) 1.63 (overlapped) 4.49 (dd, 11.7, 4.6) – 0.80 (overlapped) 1.42 (overlapped) – –
20 21 22 23 24 25 26 27 28α
9
61.7 (d)
2.35 (s)
28β
10 11 12 13 14 15 16 17 18 19
37.0 (s) 199.9 (s) 128.3 (d) 169.4 (s) 45.5 (s) 25.8 (t) 21.5 (t) 36.9 (s) 42.7 (d) 44.9 (t)
– – 5.57 (s) – – 1.63 (overlapped) – – 2.13 (dd, 10.5, 3.0) 1.72 (d, 13.5)
29 30 1′ 2′ 3′ 4′–13′ 14′ 15′ 16′
t3:3
Position
t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12
1α 1β 2 3 4 5 6 7 8
t3:13 t3:14 t3:15 t3:16 t3:17 t3:18 t3:19 t3:20 t3:21 t3:22 t3:23
δC
C
255 256
38.8 (t)
N
253 254
U
251 252
δH (J in Hz)
T
267 268
unsaturation were deduced, and the leaving one unsaturation requires one ring contained in the structure. A δ-lactone moiety was constructed from the 1H–1H COSY correlations of H-3/H-4/H-5/H-6 and HMBC correlations form H-6 to C-2 and from H-4 to C-2. The 1H–1H COSY correlations of H-5/H-7, H-4/H-8, and H-3/H-11 suggested the direct linkages of C-5 with C-7, C-4 with C-8, and C-3 with C-11. In the ROESY spectrum, the cross-peaks of H-4 with H-5 and H-11, and H-3 and H-7 with H-8 suggested the α-orientation of H-4 and H-5 and β-orientation of H-3 (Fig. 2). Thus, the structure of compound 1 was characterized as (3S, 4S, 5R)-4(2-hydroxyethyl)-3-(hydroxymethyl)-5-vinyltetrahydro2H-pyran-2-one and named as swertiachiralatone A (1). Compound 2 was deduced with the molecular formula C21H34O8 by HRESIMS (m/z 415.2343 [M + H]+, calcd. 415.2326). The IR spectrum showed the presence of hydroxyl (3423 cm− 1), double bond (1650 cm− 1) and glycosidic linkage (1076 cm− 1) in the structure. The 13C NMR (DEPT) spectrum exhibited 21 carbons, including 4 quaternary carbons, 9 methines, 5 methylenes and 3 methyl
δC
F
55.2 (d)
Position
O
1α 1β 2 3 4 5 6α 6β 7 8α 8β 9α 9β 10
δH (J in Hz)
R O
t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17
δC
P
Position
D
t2:3
E
t2:1 t2:2
N.-J. Zhou et al. / Fitoterapia xxx (2014) xxx–xxx
δC
δH (J in Hz) 31.1 (s) 31.9 (t) 30.7 (t) 28.1 (q) 16.4 (q) 16.8 (q) 18.6 (q) 23.4 (q) 69.7 (t)
33.0 (q) 23.4 (q) 173.7 (s) 34.9 (t) 33.8 (t) 29.7–29.2 (t) 23.6 (t) 22.7 (t) 14.2 (q)
– – 1.42 (overlapped) 0.87 (overlapped) 1.15 (s) 0.87 (overlapped) 1.10 (s) 1.38 (s) 3.45 (d, 10.9) 3.22, d, 12.0 3.19 (d, 10.9) 3.22, d, 12.0 0.92 (s) 0.87 (overlapped) – 2.27 (t, 7.5) – – – 1.94 (m) 0.87 (overlapped)
Please cite this article as: Zhou N-J, et al, Anti-hepatitis B virus active constituents from Swertia chirayita, Fitoterapia (2014), http:// dx.doi.org/10.1016/j.fitote.2014.11.011
269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288
N.-J. Zhou et al. / Fitoterapia xxx (2014) xxx–xxx t4:1 t4:2
5
Table 4 NMR data of swertiachoside B (4) in CD3OD (400 Hz for 1H and 400 Hz for 13C). Position
δC
δH (J in Hz)
Position
δC
δH (J in Hz)
t4:4 t4:5 t4:6 t4:7 t4:8 t4:9 t4:10 t4:11 t4:12 t4:13 t4:14
1 2 3 4 4a 4b 5 6 7α 7β 8
163.4 (s) 100.2 (d) 166.2 (s) 95.1 (d) 159.3 (s) 168.4 (s) 67.6 (d) 27.4 (t) 27.9 (t)
– 6.17 (d, 2.0) – 6.35 (d, 2.0) – – 4.58 (dd, 9.5, 7.0) 2.06 (m) 2.29 (dq, 13.3, 3.0) 1.76 (t, 13.3) 4.94 (overlapped)
8a 8b 9 1′ 2′ 3′ 4′ 5′ 6′α 6′β
117.8 (s) 105.3 (s) 183.0 (s) 105.2 (d) 71.7 (d) 78.0 (d) 75.8 (d) 78.2 (d) 62.9 (t)
– – – 4.68 (d, 7.6) 3.19 (dd, 12.0, 2.0) – – – 3.88 (dd, 12.0, 2.0) 3.69 (dd, 12.0, 2.0)
R O O
P
D E T C E R R
295
O
293 294
methyl groups and one axial proton [δH = 4.49 (1H, dd, J = 11.7, 4.6 Hz, H-3)]. The13C NMR (DEPT) data showed 46 carbon signals ascribed to 8 methyls [δC 28.1 (C-23), 16.4 (C-24), 16.8 (C-25), 18.6 (C-26), 23.4 (C-27), 33.0 (C-29), 23.4 (C-30), 14.2 (C-16′)], 24 methylenes, 5 methines and 9 quaternary carbon atoms, from which one olean-type triterpene motif and one long-chain fatty acid moiety were easily recognized. The 1H
N C
291 292
isopterocarpolone 12-O-β-D-glucopyranoside and named as swertiachoside A (2). Compound 3 had a molecular formula of C46H78O4 from HRESIMS at m/z 717.5763 [M + Na]+ (calcd for 717.5792). Its IR spectrum showed absorptional bands at 3448 and 1653 cm− 1 ascribed to hydroxyl and carbonyl functionalities. Its 1H NMR spectrum (Table 3) displayed signals of several
U
289 290
71.3 (d)
F
t4:3
Fig. 2. Key 2D NMR correlations of compounds 1–4.
Please cite this article as: Zhou N-J, et al, Anti-hepatitis B virus active constituents from Swertia chirayita, Fitoterapia (2014), http:// dx.doi.org/10.1016/j.fitote.2014.11.011
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314 315 316 317 318 319 320 321 322 323 324 325
t5:3
Compounds
HBsAgb
CC50 [mM]
b c d e f
0.18
0.043 0.36 0.44
0.076
e
C
SI
– – – – – – – – – b1 – – – 4.29 – – 0.20 – 1.23 – – – – – – – – – – – N1.39
E
R
R
0.55
N4.75 N2.04 N1.88 N2.49 N4.14 N2.15 N3.03 N1.81 N2.56 0.40 ± N0.88 N4.53 N1.98 0.31 ± N3.41 N3.79 1.48 ± N3.04 1.49 ± N3.20 N3.28 N10.49 N1.67 N0.65 N1.19 N1.00 N3.22 N7.18 N1.45 N2.68 1.25 ±
O
0.21 0.0038
C
a
0.55 0.12
N
t5:35 t5:36 t5:37 t5:38 t5:39 t5:40
N4.75 N2.04 N1.88 N2.49 3.43 ± 1.34 ± N3.03 N1.81 1.38 ± 0.02 ± N0.88 4.25 ± N1.98 1.35 ± N3.41 N3.79 0.30 ± N3.04 1.83 ± 2.73 ± N3.28 N10.49 N1.67 N0.65 N1.19 N1.00 N3.22 N7.18 0.66 ± N2.68 N1.74
U
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Tenofovirf
342
Compounds 1–30 was evaluated for their anti-HBV activity on HepG 2.2.15 cells in vitro, namely inhibiting the secretions of HBsAg and HBeAg, and HBV DNA replication using tenofovir as the positive control [22]. As shown in Table 5, compounds 10, 14, 17, 19 showed activity inhibiting HBsAg secretion; compounds 14 and 28 showed activity inhibiting HBeAg
343
HBeAgc
IC50 [mM] t5:4 t5:5 t5:6 t5:7 t5:8 t5:9 t5:10 t5:11 t5:12 t5:13 t5:14 t5:15 t5:16 t5:17 t5:18 t5:19 t5:20 t5:21 t5:22 t5:23 t5:24 t5:25 t5:26 t5:27 t5:28 t5:29 t5:30 t5:31 t5:32 t5:33 t5:34
3.2. In vitro anti-HBV activity
F
312 313
O
310 311
T
308 309
R O
Table 5 Anti-HBV activities of compounds 1–30a.
307
327 328
P
t5:1 t5:2
305 306
data (Table 2) were similar to those of tetrahydroswertianolin [50], except for the absence of the methoxy group at C-3, from which compound 4 was proposed to be the 3-O-demethylated derivative of tetrahydroswertianolin. The full NMR data assignments of compound 4 were performed with the aid of the HSQC, 1H–1H COSY and HMBC data (Fig. 2). In the HMBC spectrum, the correlation of H-1′ with C-8 indicated that the glucosyl group was attached to C-8. The cross-peaks of H-5 with H-8 in the ROESY spectrum suggested that H-5 and H-8 were located at the same side. Its absolute configuration was determined to be identical with tetrahydroswertianolin by their similar [α]D values (tetrahydroswertianolin: [α 24 D ]: +8.0, c 0.2, MeOH; compound 4: [α 24 D ]: +10.5, c 0.11, MeOH). Hence, compound 4 was defined to be 3-nortetrahydroswertianolin and named as swertiachoside B (4) as shown in Fig. 1.
E
326
and 13C NMR (DEPT) data of compound 3 were almost identical to those of 3β-hydroxy-11-oxo-olean-12-enyl-3-palmitate (24) [40], except that one methyl (C-28) in 24 was changed to hydroxymethyl group [δC 69.7 (C-28)] in 3, which was supported by the HMBC correlations of H-28 with C-16, C-17, C-18 and C-22 (Fig. 2). The palmityloxy group was proposed at C-3 position from the HMBC correlation of H-3 to C-1′. The H-3 was determined to be an α-orientation from the splitting and coupling constants (J) of the proton signal at 4.49 (dd, J = 11.7, 4.6 Hz), and the ROESY correlation of H-3 with H-23. Similarly, the ROESY correlations of H-25 with H-24 and H-26, H-9 with H-5 and H-27 indicated the α-orientation of H-5, H-9 and Me-27 and the β-orientation of Me-24, Me-25 and Me-26. Consequently, the structure of compound 3 was determined as 3β, 28-dihydroxy-11-oxo-olean-12-enyl-3-palmitate and named as swertiachirdiol A (3). Compound 4 possessed a molecular formula of C19H22O11 determined by HRESIMS at m/z 425.1081 [M – H]+ (calcd for 425.1084). The IR spectrum suggested the presence of hydroxyl (3440 cm−1) and hydrogen bonded ketone (1653 cm−1) groups. Hydrolysis of compound 4 with 2 M HCl in methanol to yield D-glucose which was identified by comparing with the authentic sample on TLC, as well as the [α]D experiment 1 13 ([α 25 C NMR (DEPT) spectral D ]: +55.7, c 0.14, H2O). Its H and
303 304
D
6
0.035
0.045
0.23 0.33
0.025
DNAd
IC50 [mM]
SI
IC50 [mM]
SI
N4.75 N2.04 N1.88 N2.49 N4.14 N2.15 N3.03 N1.81 N2.56 N1.47 N0.88 N4.53 N1.98 0.77 ± 0.076 N3.41 N3.79 N2.25 N3.04 N2.43 N3.20 N3.28 N10.49 N1.67 N0.65 N1.19 N1.00 N3.22 5.92 ± 1.02 N1.45 N2.68 1.21 ± 0.031
– – – – – – – – – – – – – 1.75 – – – – – – – – – – – – – N1.21 – – N1.44
N1.19 N0.51 N0.47 N0.62 N1.03 N0.54 N0.76 0.30 ± 0.051 0.070 ± 0.010 N0.47 N0.22 N1.13 0.16 ± 0.021 0.29 ± 0.034 N0.85 N0.95 0.33 ± 0.055 N0.76 N0.61 N0.80 N0.82 N2.62 N0.42 0.22 ± 0.045 0.13 ± 0.021 0.24 ± 0.045 N0.80 N1.80 0.22 ± 0.037 N0.67 0.0011 ± 0.00043
– – – – – – – N6.03 19.71 – – – N12.38 4.66 – – b1 – – – – – – N4.57 N5.07 N4.85 – – 2.97 – N1581.82
All values are the mean of two independent experiments. HBsAg: HBV surface antigen. HBeAg: HBV e antigen. DNA: HBV DNA replication. CC50 = 50% cytotoxic concentration, IC50 = 50% inhibition concentration, SI (selectivity index) = CC50 / IC50. Tenofovir, an antiviral agent used as a positive control.
Please cite this article as: Zhou N-J, et al, Anti-hepatitis B virus active constituents from Swertia chirayita, Fitoterapia (2014), http:// dx.doi.org/10.1016/j.fitote.2014.11.011
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369
Four new compounds, involving one unusual eudesmane sesquiterpene and one tetrahydroxanthone, together with twenty-six known compounds were firstly isolated from S. chirayita. Eleven compounds showed anti-HBV activities on HepG 2.2.15 cells line in vitro (four compounds showed activity inhibiting HBsAg secretion, two compounds showed activity inhibiting HBeAg secretion, and nine compounds exhibited activity inhibiting HBV DNA replication). This work will provide valuable information for revealing the anti-HBV active constituents of S. chirayita.
370
5. Statement
361 362 363 364 365 366 367 368
371
The authors declare no conflict of interest.
T
356 357
Acknowledgments
373 374
379
This work was supported by the International Foundation for Science in Sweden (No. F/5202-1), the National Natural Science Foundation of China for Distinguished Young Scholars (No. 81025023), the West Light Foundation of the Chinese Academy of Sciences, the National Natural Science Foundation of China (No. 81202436), and the Youth Innovation Promotion Association, CAS.
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Appendix A. Supporting information
381
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1D and 2D NMR, HREIMS/HRESIMS, IR, UV, [α]D spectra of compounds 1–4 are available as Supporting information. Supplementary data related to this article can be found online at doi: http://dx.doi.org/10.1016/j.fitote.2014.11.011
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secretion; and compounds 8, 9, 13, 14, 17, 24–26 and 29 exhibited activity inhibiting HBV DNA replication. In particular, compound 13 displayed obviously inhibition on HBV DNA replication with IC50 value of 0.16 ± 0.021 mM, with high SI value of N12.38. (+)-Cycloolivil-4′-O-β-D-glucopyranoside (14) exhibited inhibition not only on the secretions of HBsAg and HBeAg with IC50 values of 0.31 ± 0.045 mM (SI = 4.29) and 0.77 ± 0.076 mM (SI = 1.75), respectively, but also on HBV DNA replication with an IC50 value of 0.29 ± 0.034 mM (SI = 4.66).
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Please cite this article as: Zhou N-J, et al, Anti-hepatitis B virus active constituents from Swertia chirayita, Fitoterapia (2014), http:// dx.doi.org/10.1016/j.fitote.2014.11.011
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